Introduction {#S1}
============

The production of biofuels from renewable resources has gained significant attention due to the rising energy crisis and environmental concerns. Currently, bioethanol is widely used, and Grand View Research, Inc., reported that the global ethanol market size could reach \$115.65 billion by 2025, growing at a compound annual growth rate (CAGR) of 6.7%^[1](#footnote1){ref-type="fn"}^. In addition, the microbial production of higher alcohols (especially C3--C6) has gained traction over the last decade. The use of higher alcohols such as isopropanol or isobutanol would not require changes to current biofuel refinery or transportation processes as these alcohols can be blended at higher volumes in gasoline compared to ethanol (e.g., 16% for isopropanol/isobutanol versus 10% for ethanol), resulting in lower greenhouse gas (GHG) emissions ([@B3]; [@B55]). However, higher alcohols, except n-butanol, are not commonly produced at high yields in microbes. With the development of molecular biology techniques and metabolic engineering strategies, model systems, such as *Escherichia coli* ([@B27]; [@B24]; [@B33]; [@B42]) and *Saccharomyces cerevisiae* ([@B46]; [@B52]), have been modified to synthesize bioalcohols.

*Escherichia coli* is a well-studied model microorganism which has several advantageous traits for bioalcohol production including fast growth in inexpensive mineral media, the ability to utilize a wide range of substrates from biomass, and detailed genetic information and diverse genetic tools for gene manipulation. However, there are still challenges using engineered *E. coli* for industrial applications such as the need to improve tolerance to bioalcohols, efficient utilization of low-cost substrates, and productivity toward advanced alcohols ([@B27]; [@B24]; [@B33]; [@B42]). Recently, the rapid expansion of genome engineering strategies, synthetic biology techniques, and high-throughput tools have enabled their application to study advanced bioalcohols production and to further investigate the mechanisms of alcohol resistance. This review will summarize recent progress in metabolic engineering of *E*. *coli* for C2--C6 alcohol-derived biofuel production ([Figure 1](#F1){ref-type="fig"}), introduce new synthetic biology methods and genome engineering strategies for in-depth studies of alcohol tolerance, and analyze the current challenges for increasing productivity for industrial applications.

![The metabolic pathways for the production of C2--C6 bioalcohol in *E. coli*. The genes directly related to bioalcohol synthesis are labeled in different colors. C2 (red); C3 (orange); C4 (green); C5 (blue); C6 (purple). The substrates are labeled in red. Relevant reactions are represented by the name of the gene(s) coding for the enzyme(s): *adh2/adhE*, alcohol dehydrogenase; *alsS*, acetolactate synthase; *atoB*, acetyl-CoA acyltransferase; *cimA*, citramalate synthase; *dxr*, 1-deoxy-[D]{.smallcaps}-xylulose 5-phosphate reductoisomerase; *dxs*, 1-deoxy-[D]{.smallcaps}-xylulose 5-phosphate synthase; *HMGR*, 3-hydroxy-3-methylglutaryl-CoA reductase; *HMGS*, 3-hydroxy-3-methylglutaryl-CoA synthase; *idi*, isopentenylpyrophosphate isomerase; *ilvA*, threonine deaminase; *ilvC*, acetohydroxy acid isomeroreductase; *ilvD*, dihydroxy acid dehydratase; *ilvGM*, acetohydroxybutanoate synthase; *ispD*, 4-diphosphocytidyl-2-methylerythritol synthase; *ispE*, 4-diphosphocytidyl-2-methylerythritol kinase; *ispF*, 2-methylerythritol 2,4-cyclodiphosphate synthase; *ispG*, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase; *ispH*, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase; *kivd*, ketoisovalerate decarboxylase; *leuA*, 2-isopropylmalate synthase; *leuB*, 3-isopropylmalate dehydrogenase; *leuCD*, 2-isopropylmalate isomerase; *MK*, mevalonate kinase; *nudF*, prenyl phosphatase; *PMD*, phosphomevalonate decarboxylase; *PMK*, phosphomevalonate kinase; *DHAP*, dihydroxyacetone-phosphate; *DMAPP*, dimethylallyl pyrophosphate; *Gly-3-P*, glyceraldehyde-3-phosphate; *IPP*, isopentenyl pyrophosphate; *PEP*, phosphoenolpyruvate.](fbioe-08-00710-g001){#F1}

Ethanol (C2 Bioalcohol) Production in *E. Coli* {#S2}
===============================================

The current chassis for industrial ethanol production is *S. cerevisiae* due to its ability to produce ethanol from glucose at 95% maximum theoretical yield ([@B32]). The bacterium *Zymomonas mobilis* has also been proposed for use in industrial ethanol production since it also produces ethanol from glucose at 95% of maximum theoretical yield and has a higher specific ethanol productivity than *S. cerevisiae* ([@B77]). Next generation biofuels and biochemicals aim to use lignocellulosic biomass, which contains both glucose and xylose, as an attractive source of non-food carbohydrates for production ([Table 1](#T1){ref-type="table"}) ([@B67]). However, neither *S. cerevisiae* nor *Z. mobilis* natively utilize xylose. In order to produce bioethanol from cellulosic feedstocks, the cellulose-degrading bacterium *Clostridium thermocellum* has also been used as a chassis for industrial production because it is able to directly ferment cellulose into ethanol. However, *C. thermocellum* only generates ethanol from cellulose (and also only natively utilizes glucose) at 75% of the maximum theoretical yield, resulting in lower ethanol yields compared to *S. cerevisiae* or *Z. mobilis* ([@B61]). Furthermore, *C. thermocellum* has low tolerance to ethanol ([@B22]) and has limited genetic tools, making it difficult and time consuming to engineer this strain for increased ethanol production ([@B63]), although CRISPR-Cas-based genome editing systems were recently developed ([@B64]).

###### 

Select examples of engineering *E. coli* for bioalcohol production.

  ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  Alcohol                                     General strategy                                                                                                                                                                    Method                                                                                                                                                                                                                                      Titer (g/L)                  Yield (g/g)    References
  ------------------------------------------- ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ---------------------------- -------------- ------------
  Ethanol                                     Improve substrate utilization                                                                                                                                                       \(i\) Ethanologenic *E. coli* strains carry the *Z. mobilis pdc*, *adhA*, and *adhE* genes\                                                                                                                                                 46                           0.45           [@B67]
                                                                                                                                                                                                                                  (ii) Deletion of *xylR* gene to make the LYglc1 strain\                                                                                                                                                                                                                                 
                                                                                                                                                                                                                                  (iii) Deletion of *ptsI*, *ptsG*, *galP*, and *glk*; expression of XylR\* to make the LYxyl3 strain\                                                                                                                                                                                    
                                                                                                                                                                                                                                  (iv) Co-culture of LYglc1 and LYxyl3 strains                                                                                                                                                                                                                                            

  Isopropanol                                 Improve precursor accumulation; decrease the metabolic flux to TCA                                                                                                                  Construction of BW25113 (Δ*lacI*, Δ*gltA*) with plasmid pTA17 (*P*~L~lacO~1~::*thl*, *atoAB*, *adc*, and *adhE*), pTA965 (*P*~L~lacO~1~::*tetR*, *P*~L~tetO~1~::*gltA*.LAA), and pTA1251 (*P*~L~lacO~1~::*poxB*, *acs*, *P*lacIq::*lacI*)   3.8                          Not reported   [@B57]

  Isobutanol                                  Directed evolution with alcohol-biosensor-based selection                                                                                                                           \(i\) BmoR-based biosensor used in an atmospheric and room temperature plasma (ARTP) mutagenesis library to screen for increased isobutanol production\                                                                                     56.6                         Not reported   [@B75]
                                                                                                                                                                                                                                  (ii) Fed-batch fermentation with gas-stripping                                                                                                                                                                                                                                          

  n-butanol                                   Inactivation of byproduct pathway; improve substrate utilization; improve cofactor supply; adaptive evolution for improved cell growth; optimize the expression of pathway genes\   \(i\) Gene knockout of *hyc-hyp*, *fdhF*, *poxB*, *pck*, *fumB*, *fumAC*, *tdcD*, *mdh*, *focA*, *ppc*, *mgsA*, *yieP*, *stpA*, *yqeG*, *yagM* in BW25113\                                                                                  20                           0.34           [@B13]
                                                                                                                                                                                                                                  (ii) Integration of the *fdh* gene into the genome with P~ydfZ~\                                                                                                                                                                                                                        
                                                                                                                                                                                                                                  (iii) Adaptive evolution for fast anaerobic cell growth\                                                                                                                                                                                                                                
                                                                                                                                                                                                                                  (iv) RBS library for the *phaA*, *hbd*, *crt*, *ter*, and *adhE2* genes                                                                                                                                                                                                                 

  2-methyl-1-butanol and 3-methyl-1-butanol   Inactivation of byproduct pathway; improve substrate utilization; optimize the expression of pathway genes\                                                                         \(i\) Construction of *E. coli* AY3 (BW25113, Δ*glnA*, Δ*gdhA*, Δ*lsrA*, pYX68 (*ilvE-ilvA-sdaB*), pYX90 (*alsS-ilvC-ilvD-avtA*), pYX97 (leuDH-kivd-yqhD)\                                                                                  2.2 (two alcohols mixture)   Not reported   [@B36]
                                                                                                                                                                                                                                  (ii) Construction of *E. coli* BLF2 \[*E. coli* B Δ*ldh*, pLF101 (*alsS-ilvC-ilvD*), pLF102 (*kivd-yqhD*)\]\                                                                                                                                                                            
                                                                                                                                                                                                                                  (iii) Co-culture of AY3 and BLF2 with an inoculation ratio of 1:4 using distillers' grains with solubles\                                                                                                                                                                               

  Pentanol                                    Inactivation of byproduct pathway; optimize the expression of pathway genes\                                                                                                        \(i\) Construction of BW25113 (Δ*ilvB* Δ*ilvI* Δ*leuA*) transformed with plasmid pAFC52 (*cimA*Δ2, *leuBCD*) and pGC22 \[*leuA* (G462D), *kivd* (V461G), *yqhD*\]\                                                                          4.3                          Not reported   [@B9]
                                                                                                                                                                                                                                  (ii) *In situ* extraction using oleyl alcohol                                                                                                                                                                                                                                           

  Isoprenol                                   Improve precursor accumulation; optimize the expression of pathway genes\                                                                                                           \(i\) Construction of AK26 (*E. coli* DH1), transformed with plasmids JBEI-17081 (pA5c-AtoB-HMGS_Sa-HMGR_Sa) and JBEI-17844 (pTrc99a-PMDsc_HKQ-MKmm) (ii) Fed-batch cultures with a solvent overlay                                         10.8                         0.105          [@B30]
  ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

*Escherichia coli* is additionally investigated as a host for ethanol production because it has a large variety of metabolic engineering tools available for strain modification, it can grow in higher concentrations of ethanol ([@B76]), and it naturally ferments both glucose and xylose, although the presence of glucose still leads to carbon catabolite repression (CCR) when xylose is present ([@B38]). Thus, [@B15] developed an *E. coli* co-culture strategy for conversion of glucose-xylose mixtures to ethanol. One strain, LYglc1, was engineered to only utilize glucose by deleting the xylose-specific transcriptional activator, XylR. The other strain, LYxyl3, was engineered to only utilize xylose by mutating XylR to remove CCR and by deleting genes (Δ*ptsI* Δ*ptsG* Δ*galP glk::kanR*) required for glucose transport and metabolism. The strains are ethanologenic due to insertion of the *pdc*, *adhA*, and *adhE* genes from *Z. mobilis*. The LYglc1 and LYxyl3 strains were co-cultured at an optimum ratio of 1:500, enhancing the sugar utilization rate and ethanol productivity by 50 and 28%, respectively, when compared to a monoculture of the parent strain, LY180. Using this system, they achieved productivity of 0.49 g/L/h, with a final ethanol titer of 46 g/L at 90% of maximum theoretical yield. In a different approach, [@B59] constructed a "two-phase-two-temperature" strategy using temperature inducible promoters to control the glucose metabolic pathway in *E. coli*. The final strain, B0013-2021HPA (Δ*ptsG* Δ*manZ* Δ*glk*; *ptsG* expressed under the control of tandem λ pL and pR promoters), utilized all sugars but glucose for cell growth at 34°C, whereas it fermented all sugars to ethanol at 42°C. In addition, *Z. mobilis pdc* and *adhB* genes were introduced to increase ethanol production. As a result, this strain produced 127 g ethanol from 260.9 g mixed sugars from corncob hydrolysate with a productivity of 4.06 g/L/h.

In addition to strategies to improve ethanol yield, there have been several studies to understand ethanol-induced stress and improve ethanol tolerance in *E. coli* ([@B39]; [@B8]; [@B41]). [@B8] systematically analyzed the mechanism for ethanol-induced stress and found that ethanol damages cell wall and membrane integrity, decreases the cross-membrane proton gradient and related ATP synthesis, and changes protein functions by direct binding. Genes that are upregulated in response to ethanol stress include: *osmBC* and *ompCGLR* in response to osmotic stress; *gadABE* and *asr* in response to acid stress; *rpoE*, *degP*, *asnB*, and *opgG* in response to envelope stress; *groSL*, *grpE*, and *metA* in response to heat-shock stress; and the OxyR and SoxRS regulons in response to ROS. Therefore, these cellular processes and genes are good targets for engineering strains with increased ethanol tolerance. Expression of heterologous genes can also lead to increased ethanol tolerance. [@B39] expressed the *yajC* gene, which encodes a subunit of a protein translocase complex, from *Lactobacillus buchneri* in *E. coli*, and increased tolerance up to 4% ethanol.

C3--C4 Alcohol Tolerance and Production in *E. Coli* {#S3}
====================================================

C3--C4 alcohols such as propanol, isopropanol, 1-butanol, and isobutanol are higher alcohols which have similar fuel properties ([@B11]). Several species of *Clostridium* have been evaluated for butanol and isopropanol production, but cannot be used for industrial application mainly due to low fermentation yield and titer ([@B60]; [@B73]). Alternative organisms such as *E. coli* have also been engineered toward the goal of industrial production of C3--C4 alcohols ([@B27]; [@B24]; [@B33]; [@B42]). *E. coli* metabolic pathways have shared intermediate metabolites, which reduce central metabolites such as acetyl-CoA and pyruvate into more electron-rich compounds and higher carbon acyl-CoA and 2-keto acids ([@B49]; [@B21]; [@B45]; [@B57]; [@B44]).

Precursor accumulation is one of the limiting steps for isopropanol production ([@B11]; [@B57]). The *gltA* gene is involved in synthesizing isocitrate from oxaloacetate. [@B56] developed a metabolic toggle switch (MTS) method by expressing the *gltA* gene under the P~*L*~tetO~1~ promoter and the TetR repressor under the P~*L*~lacO~1~ promoter; this system allows metabolic flux from the TCA cycle to be redirected toward isopropanol production in an inducible manner. Then, they introduced a plasmid that overexpressed the native *poxB* and *acs* genes under the P~*L*~lacO~1~ promoter for conversion of excess pyruvate to acetyl-CoA ([@B57]). The resulting isopropanol titer was up to 3.8 g/L, a titer 4.4-fold higher than that of the parent strain. Our group applied a CRISPR-based genome engineering strategy to generate ribosome binding site (RBS) libraries (903 mutants in total) for genes in the synthetic pathway for the production of isopropanol (*thl*, *atoDA*, *adc*, and *adh*), and were able to identify a high isopropanol producer, PA14, that generated 7.1 g/L at 24 h, with a yield of 0.75 mol/mol glucose. In particular, we found that higher expression levels of *adc* and *adh* led to increased isopropanol production ([@B34]).

Other efforts have focused on producing bioalcohols from alternate carbon sources. To enable utilization of acetate as the sole carbon source for isopropanol production, [@B74] first constructed the isopropanol pathway by combining genes from *Clostridium acetobutylicum* (*thlA*, *adc*), *E. coli* (*atoDA*), and *Clostridium beijerinckii* (*adh*). In addition, they replaced the promoter of the native *ack-pta* genes to improve the acetate kinase and phosphotransacetylase (ACK-PTA) pathway and overexpressed the native *nadK* gene to increase NADH supply. The highest concentration and yield of isopropanol reached was 1.47 g/L and 0.56 g/g acetate. Other efforts have also focused on improving tolerance to isopropanol. [@B23] performed adaptive laboratory evolution (ALE) in *E. coli* and identified five mutations (*relA*, *marC*, *proQ*, *yfgO*, and *rraA*) with enhanced isopropanol tolerance up to 27 g/L. Transcriptome analysis revealed that genes related to amino acid biosynthesis, iron homeostasis, and energy metabolisms are related to isopropanol tolerance. [@B78] used a targeted deletion approach and demonstrated that isopropanol tolerance could be increased by inactivation of the acetoacetyl-CoA transferase genes and *atoDA*, enabling growth in 500 mM isopropanol.

Improved fermentative production of isobutanol, a non-native alcohol pathway, has been achieved by metabolic engineering approaches in *E. coli* strains ([@B7]). Recently, [@B58] overexpressed the native *acs*, *pckA*, and *maeB* genes to increase acetate uptake, resulting in 26% increased isobutanol titers using acetate as the sole carbon source. Other efforts have focused on increasing isobutanol production from glucose. [@B35] introduced the heterologous Entner--Doudoroff (ED) pathway from *Z. mobilis* to increase glucose transformation to pyruvate for enhanced precursor accumulation. The resulting *E. coli* strain, ED02, produced 13.67 g/L isobutanol with a productivity of 0.456 g/L/h, a 56.8 and 88.1% improvement over the parent strain, respectively. [@B18] used the Optimization by Selection and Sequencing (OptSSeq) strategy to regulate expression levels of genes in the isobutanol synthetic pathway. They found that the optimum levels of pathway enzymes (AlsS, IlvC, IlvD, Kivd, and AdhA) were a molar ratio of 2.5:6.7:2:1:5.2, which led to 3 g/h/gDCW of isobutanol production. In addition, [@B75] used a BmoR-based biosensor to screen for improved isobutanol producing strains from an atmospheric and room temperature plasma (ARTP) mutagenesis library. The best isolated variant produced twofold more isobutanol than the wild-type, and the titer of isobutanol reached 56.5 g/L, with a productivity of 0.533 g/L/h, during fed-batch fermentation.

*Clostridium* species have long been employed for n-butanol production through their acetone--butanol--ethanol (ABE) pathway ([@B73]). However, due to the lack of available genetic tools for *Clostridia*, these species are currently not robust candidates as industrial chassis ([@B1]). Therefore, in recent years, many efforts have been applied toward engineering *E. coli* for n-butanol production ([@B12]). Over the last 5 years, the utilization of renewable and cheap substrates from agricultural residues and crude glycerol waste streams have been a target for n-butanol production studies in *E. coli*. [@B1] integrated butanol pathway genes (*hbd*, *crt*, *adhE2*, *ter*, and *atoB*) into xylose-utilizing host SSK42 (*E. coli* B P~gapA~PDH Δ*ldhA* Δ*frdA* Δ*pfB*), and the final strain produced 4.3 g/L butanol using xylose as the sole carbon source. Similarly, [@B48], [@B50] first integrated butanol pathway genes (*phaA*, *hbd*, *crt*, *ter*, and *adhE2*) into a BL21-based host strain with byproduct gene deletions (Δ*ptsG*, Δ*poxB1*, Δ*ldhA*, Δ*frdA*, and Δ*adhE*) for glycerol conversion to butanol. In addition, they increased NADH regeneration by overexpression of genes related to NADH-production (*aceEF*, *lpdA*, *zwf*, *pgl*, and *udhA*). As a result, the engineered strain produced 6.9 g/L n-butanol from 20 g/L crude glycerol under microaerobic conditions, increasing productivity fivefold compared to the strain without modification of NADH supply.

Maintaining cofactor balance and resolving free CoA imbalance are important for CoA-dependent n-butanol production ([@B44]). To this end, various groups have overexpressed formate dehydrogenase (Fdh) for NADH regeneration under endogenous fermentation regulatory elements (FREs) control ([@B71]), knocked out the *pgi* gene for increased NADH by activation of the pentose phosphate pathway (PPP) ([@B49]), decreased carbon flux from acetyl-CoA to the TCA cycle, and improved NADH and CoA supply by downregulation of citrate synthase ([@B49]; [@B21]), knocked out genes from the glyoxylate shunt for increased CoA accumulation ([@B44]), and optimized AdhE2 activity for CoA recycling and supplemented with cysteine for increased CoA supply ([@B45]). All of these approaches resulted in increased n-butanol production. In addition, [@B13] developed a completely chromosomally engineered *E. coli* strain capable of producing butanol efficiently ([Table 1](#T1){ref-type="table"}). They first integrated the butanol pathway genes into a BW25113-based host strain with deleted byproduct genes. They then modified the expression of *fdh* to increase NADH regeneration and improve anaerobic cell growth by adaptive evolution. The final strain, which also had an optimized butanol pathway (from RBS libraries for pathway genes), produced 20 g/L n-butanol at 83% of theoretical yield, the highest titer achieved compared to the above studies. Low n-butanol tolerance is also limiting for the economic viability of n-butanol production. A number of studies have identified strategies that improve tolerance: (1) mutation of genes related to cis-regulatory elements (*yqjA*, *yabI*, and *rob*) or the efflux pump subunit, acr*B* ([@B26]; [@B20]); (2) disruption of the transmembrane protein, TqsA ([@B20]); (3) disruption of succinylglutamate desuccinylase (AstE) ([@B19]); (4) overexpression of the chaperone protein SecB or its mutation, SecB~T10A~ ([@B72]); and (5) overexpression of the membrane-targeted tilapia metallothionein, OmpC-TMT ([@B10]).

C5--C6 Alcohol Production and Tolerance in *E. Coli* {#S4}
====================================================

C5 alcohols such as 1-pentanol, 2-methyl-1-butanol, and 3-methyl-1-butanol (isopentanol) and C6 alcohols such as 1-hexanol, 3-methyl-1-pentanol, and 4-methyl-1-pentanol represent a useful class of chemicals with potential application as biofuels ([@B66]). In *E. coli*, iterative keto-acid elongation resulted in C5, C6, and even longer chain (C7--C8) alcohols ([@B66]). Recently, [@B14] constructed a new isopentanol production pathway in *E. coli* XX03 (BW25113 Δ*adhE* Δ*ldhA* Δ*frdBC*) by introducing the isovaleryl-CoA pathway (LiuC, AibAB, and AibC) from *Myxococcus xanthus* and a butyryl-CoA reductase (AdhE2) from *C. acetobutylicum*, and the resulting strain produced 80.5 mg/L isopentanol after 36 h under microaerobic conditions. In addition, [@B9] constructed a 1-pentanol production pathway by controlling the keto acid elongation cycle \[BW25113 Δ*ilvB* Δ*ilvI* Δ*leuA* expressing *cimA*Δ2, *leuBCD*, *leuA* (G462D), *kivd* (V461G), *yqhD*\] and identifying a new mutation in the ketoisovalerate decarboxylase, KivD V461D, that preferentially tuned the KivD from *Lactococcus lactis* specificity toward 1-pentanol synthesis. The titer of 1-pentanol reached 4.3 g/L and comprised 90% of the total alcohol content. However, the titer, yield, and productivities for C5-C6 bioalcohol production in *E. coli* are still too low for industrial applications. Recently, Chen and coworkers employed an adaptive evolution method to increase the tolerance of *E. coli* to isopropanol, isobutanol, and isopentanol, identifying that upregulated RpoS can increase general alcohol resistance ([@B65]), a strategy that may also be relevant for tolerance to other C5--C6 alcohols.

The biosynthesis of isopentenols, including isoprenol and prenol, provides an additional route to the production of C5 alcohols ([@B17]). Unlike other C5--C6 alcohols, isopentenols are synthesized from the isoprenoid pathway precursor metabolites isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) ([Figure 1](#F1){ref-type="fig"}). To improve the availability of IPP/DMAPP, [@B62] constructed a CRISPRi-mediated multiplex repression system to knockdown genes *asnA*, *gldA*, and *prpE* which are involved in asparagine production, glycerol utilization, and propionyl-CoA synthesis, respectively, resulting in 18-24% higher isopentenol. [@B28] developed "IPP-bypass" mevalonate pathways using mevalonate diphosphate decarboxylase (PMD) and phosphatase (AphA) for isopentenol production. They then further improved PMD activity through high-throughput enzyme screening ([@B29]) and optimized the origin and expression level of genes in this IPP-bypass pathway ([@B30]). Their final engineered strain had an isoprenol titer of 10.8 g/L in a fed-batch fermentation ([@B30]). Other efforts have focused on producing isoprenol from cellulosic feedstocks. [@B70] initially utilized switchgrass hydrolysate derived from ionic liquid pretreatment for isoprenol production, but the remaining ionic liquids in the hydrolysate required multiple washes to decrease its toxicity to *E. coli*. Thus, they used an adaptive evolution strategy to improve tolerance to ionic liquids, and the adapted *E. coli* strain produced 1.06 g/L which is 6.6-fold more isoprenol compared to the parent strain in the presence of ionic liquids ([@B69]). In addition, they found that NaCl enhanced the tolerance of *E. coli* to ionic liquids. MG1655 reached an OD~600~ that was ∼twofold higher with 200 mM NaCl than without NaCl ([@B68]).

Conclusion and Perspectives {#S5}
===========================

Converting renewable biomass into biofuel using engineered microbial cell factories provides a promising alternative to fossil fuels. To date, synthetic pathways have been constructed for the production of bioalcohols ranging from C2 up to C10 ([@B25]; [@B31]). The titer, yield, and productivity for C3--C6 bioalcohol production in engineered *E. coli* have improved in recent years; however, the industrial application for the production of these bioalcohols still has some way to go compared to ethanol production. Despite these limitations, some companies (e.g., Butamax and Gevo) have begun to approach commercialization of isobutanol bioproduction. However, additional efforts are still required to overcome technological limitations. New strategies for engineering current microbes still need to be identified or developed for: (1) utilization of non-food-based carbon sources, (2) tolerance to inhibitors derived from biomass or the fermentation process, and (3) tolerance to high concentrations of substrate and biofuel products ([@B6]; [@B51]). While it might be possible to engineer a single microbe for industrial use, microbial consortia could be another solution to improve biofuel production. Cultures containing multiple microbes could improve tolerance to different inhibitors and utilize various components from complex carbon sources, further improving the economics of biofuel production ([@B4]; [@B53]). In addition, feedstock cost is almost half the cost of the fermentation process ([@B5]), so developing strains that can utilize cheaper feedstocks would have a large economic benefit for industrial biofuel production. Moreover, development of new fermentation processes that increase productivity at the industrial level, such as low-cost and efficient *in situ* product removal (ISPR), could be a new requirement for material and process engineering.

Advances in technologies for DNA synthesis and sequencing have simplified the process of reprogramming metabolism for optimal production of desired chemicals. CRISPR-based technologies have increased the accuracy and speed of gene editing and regulation at genome-scale ([@B47]; [@B16]; [@B54]; [@B37]; [@B40]). In addition, advanced systems biology tools including genomics, transcriptomics, proteomics, metabolomics, and fluxomics will help facilitate the characterization, analysis, and design of new metabolic pathways for bioalcohol production ([@B2]; [@B43]). Using advanced technologies to gain a deeper understanding of tolerance mechanisms and to refine metabolism for increased titer, yield, and productivity of alcohols from various feedstocks will enable economically viable production of bioalcohols from microbial hosts such as *E. coli*, with findings from *E. coli* providing valuable insight into improving these systems in other non-model microbial chassis.

Author Contributions {#S6}
====================

All authors contributed to the analysis of the literature, compiling the related data, and writing the manuscript.

Conflict of Interest {#conf1}
====================

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

**Funding.** This project was sponsored by the U.S. Department of Energy (Grant DE-SC0018368) and the Center for Bioenergy Innovation a U.S. Department of Energy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science.

<https://www.grandviewresearch.com/press-release/global-fuel-ethanol-market>

[^1]: Edited by: Shihui Yang, Hubei University, China

[^2]: Reviewed by: Vijai Singh, Indrashil University, India; Simon Congqiang Zhang, Agency for Science, Technology and Research (A^∗^STAR), Singapore

[^3]: ^†^These authors have contributed equally to this work

[^4]: This article was submitted to Synthetic Biology, a section of the journal Frontiers in Bioengineering and Biotechnology
