Converting Escherichia coli to a Synthetic Methylotroph Growing Solely on Methanol.

Methanol, being electron rich and derivable from methane or CO2, is a potentially renewable one-carbon (C1) feedstock for microorganisms. Although the ribulose monophosphate (RuMP) cycle used by methylotrophs to assimilate methanol differs from the typical sugar metabolism by only three enzymes, turning a non-methylotrophic organism to a synthetic methylotroph that grows to a high cell density has been challenging. Here we reprogrammed E. coli using metabolic robustness criteria followed by laboratory evolution to establish a strain that can efficiently utilize methanol as the sole carbon source. This synthetic methylotroph alleviated a so far uncharacterized hurdle, DNA-protein crosslinking (DPC), by insertion sequence (IS)-mediated copy number variations (CNVs) and balanced the metabolic flux by mutations. Being capable of growing at a rate comparable with natural methylotrophs in a wide range of methanol concentrations, this synthetic methylotrophic strain illustrates genome editing and evolution for microbial tropism changes and expands the scope of biological C1 conversion.

Escherichia coli as a host for metabolic engineering.

Over the past century, Escherichia coli has become one of the best studied organisms on earth. Features such as genetic tractability, favorable growth conditions, well characterized biochemistry and physiology, and availability of versatile genetic manipulation tools make E. coli an ideal platform host for development of industrially viable productions. In this review, we discuss the physiological attributes of E. coli that are most relevant for metabolic engineering, as well as emerging techniques that enable efficient phenotype construction. Further, we summarize the large number of native and non-native products that have been synthesized by E. coli, and address some of the future challenges in broadening substrate range and fighting phage infection.

Synthetic methanol auxotrophy of Escherichia coli for methanol-dependent growth and production.

Methanol is a potentially attractive substrate for bioproduction of chemicals because of the abundance of natural gas and biogas-derived methane. To move towards utilizing methanol as a sole carbon source, here we engineer an Escherichia coli strain to couple methanol utilization with growth on five-carbon (C5) sugars. By deleting essential genes in the pentose phosphate pathway for pentose utilization and expressing heterologous enzymes from the ribulose-monophosphate (RuMP) pathway, we constructed a strain that cannot grow on xylose or ribose minimal media unless methanol is utilized, creating a phenotype termed "synthetic methanol auxotrophy". Our best strains were able to utilize methanol for growth at a rate of 0.17 ±â€¯0.006 (h-1) with methanol and xylose co-assimilation at a molar ratio of approximately 1:1. Genome sequencing and reversion of mutations indicated that mutations on genes encoding for adenylate cyclase (cyaA) and the formaldehyde detoxification operon (frmRAB) were necessary for the growth phenotype. The methanol auxotrophic strain was further engineered to produce ethanol or 1-butanol to final titers of 4.6 g/L and 2.0 g/L, respectively. 13C tracing showed that 43% and 71% of ethanol and 1-butanol produced had labeled carbon derived from methanol, respectively.

Evolutionary engineering of methylotrophic E. coli enables fast growth on methanol.

As methanol can be derived from either CO2 or methane, methanol economy can play an important role in combating climate change. In this scenario, rapid utilization of methanol by an industrial microorganism is the first and crucial step for efficient utilization of the C1 feedstock chemical. Here, we report the development of a methylotrophic E. coli strain with a doubling time of 3.5 hours under optimal conditions, comparable or faster than native model methylotrophs Methylorubrum extorquens AM1 (Td~4hr) and Bacillus methanolicus at 37°C (Td~5hr). To accomplish this, we develop a bacterial artificial chromosome (BAC) with dynamic copy number variation (CNV) to facilitate overcoming the formaldehyde-induced DNA-protein cross-linking (DPC) problem in the evolution process. We track the genome variations of 75 cultures along the evolution process by next-generation sequencing, and identified the features of the fast-growing strain. After stabilization, the final strain (SM8) grows to 20 g/L of cell mass within 77 hrs in a bioreactor. This study illustrates the potential of dynamic CNV as an evolution tool and synthetic methylotrophs as a platform for sustainable biotechnological applications.

An ATP-sensitive phosphoketolase regulates carbon fixation in cyanobacteria.

Regulation of CO2 fixation in cyanobacteria is important both for the organism and global carbon balance. Here we show that phosphoketolase in Synechococcus elongatus PCC7942 (SeXPK) possesses a distinct ATP-sensing mechanism, where a drop in ATP level allows SeXPK to divert precursors of the RuBisCO substrate away from the Calvin-Benson-Bassham cycle. Deleting the SeXPK gene increased CO2 fixation particularly during light-dark transitions. In high-density cultures, the Δxpk strain showed a 60% increase in carbon fixation and unexpectedly resulted in sucrose secretion without any pathway engineering. Using cryo-EM analysis, we discovered that these functions were enabled by a unique allosteric regulatory site involving two subunits jointly binding two ATP, which constantly suppresses the activity of SeXPK until the ATP level drops. This magnesium-independent ATP allosteric site is present in many species across all three domains of life, where it may also play important regulatory functions.

Engineering a Thermostable Keto Acid Decarboxylase Using Directed Evolution and Computationally Directed Protein Design.

Keto acid decarboxylase (Kdc) is a key enzyme in producing keto acid derived higher alcohols, like isobutanol. The most active Kdc's are found in mesophiles; the only reported Kdc activity in thermophiles is 2 orders of magnitude less active. Therefore, the thermostability of mesophilic Kdc limits isobutanol production temperature. Here, we report development of a thermostable 2-ketoisovalerate decarboxylase (Kivd) with 10.5-fold increased residual activity after 1h preincubation at 60 °C. Starting with mesophilic Lactococcus lactis Kivd, a library was generated using random mutagenesis and approximately 8,000 independent variants were screened. The top single-mutation variants were recombined. To further improve thermostability, 16 designs built using Rosetta Comparative Modeling were screened and the most active was recombined to form our best variant, LLM4. Compared to wild-type Kivd, a 13 °C increase in melting temperature and over 4-fold increase in half-life at 60 °C were observed. LLM4 will be useful for keto acid derived alcohol production in lignocellulosic thermophiles.

Investigation of B-Z transitions with DNA oligonucleotides containing 8-methylguanine.

Among various Z-form DNA inducers, such as transition metal complexes, polyamines and high ionic concentrations, 8-methylguanine have received attention as efficient chemical modifications. Although it is clear that m8-modified guanine base markedly stabilizes the Z conformation of short oligonucleotides under physiological salt conditions, how sequence composition affects the preference of Z-DNA is still not well established. In this study, various oligomers of d(CG)n or d(GC)n containing either 8-methylguanine in a different position were synthesized and their capacity of stabilizing Z-DNA were evaluated by CD spectra and then compared with each other. It is was found out that the Z-DNA stabilizing effect depend on the order of arrangement of m(8)G and m(8)rG in DNA strands and the center position is the most effective to stabilize the Z-DNA and promote the B to Z transition.