Farming opioids from sugar

Metabolic engineering of yeast cells to produce pharmaceutical and natural products has been around for some time, notably used for production of a precursor metabolite of the widely used antimalarial drug artemisinin. Galanie et al. [1] demonstrated that metabolic pathways from other organisms can be reconstructed in yeast to synthesize complex natural products such as opiates and their opioid derivatives. Opioids are widely used as painkillers and also have diverse effects on the human body. The authors engineered yeast strains to express and knockout more than 20 heterologous genes, from organisms like plants, rats, bacteria and even yeast to convert central metabolites to thebaine, a precursor for codeine – the most commonly used opiate. Biosynthetic production of natural products is tackled by modularizing the genetic circuits to produce intermediate metabolites. The authors (i) first created a module to produce (S)-reticuline from tyrosine; (ii) convert (S)-reticuline to (R)-reticuline, a key step in the pathway; (iii) optimized the conversion of (R)-reticuline to thebaine. Enhancing the production efficiency by diverting metabolic flux through the existing modules and additional pathway engineering could scale-up the process and make it possible for commercial production.

  1. Galanie S, Thodey K, Trenchard IJ, Filsinger Interrante M, Smolke CD. Complete biosynthesis of opioids in yeast. Science. 2015 Sep 4;349(6252):1095-100.

Replacing yeast genes with human genes

Consider a yeast cell and a human cell, what will happen if the genes that are essential for survival of yeast are substituted by their human orthologs? Coding sequences of these genes have diverged almost a billion years ago, but have their functions? Kachroo et. al. [1] found almost 50% of essential genes, out of nearly 400 chosen for study, can be replaced. To the question of what factors determine replaceability, they found although sequence similarity explains some aspect of it, the major determinant seems to be the biological pathways to which these genes belong. Genes making protein products having enzymatic role in metabolism, sterol biosynthesis and part of the proteasome are more amenable to humanization compared to genes involved in DNA replication, repair or cell growth. This might indicate that the machinery for some biological processes/modules have evolved very differently in the two organisms, whereas some of them have been kept as they are. What could be the reasons for slower evolution of those biological modules?

  1. Kachroo AH, Laurent JM, Yellman CM, Meyer AG, Wilke CO, Marcotte EM.
    Evolution. Systematic humanization of yeast genes reveals conserved functions and genetic modularity. Science. 2015 May 22; 348(6237):921-5.

Can we drive malaria away with gene drives?

The dreadful effects of malaria need no recalling. One strategy to counter it is by creating mutant mosquitoes that are resistant to the parasites. However, the bottleneck has always been to ensure that the mutant mosquitoes should also spread in the wild population rapidly. Gantz et. al. [1] exploited the genome editing power of CRISPR to create a method for mutagenic chain reaction (MCR) [2] in mosquitoes. MCR technology allows us to create homozygous loss-of-function mutations in the germ line of the host organism that spreads rapidly through its offspring. Inserting mutations in two genes that cause resistance to the malarial parasite, the authors engineered a gene drive that passed on the modified homozygous genes to 99% of their offspring. Gene drives based on CRISPR-Cas9 have the potential to rapidly spread through the wild population. However, a lot of work is still required to assess its stability when introduced in a gene pool and developing human regulatory control before the powerful technology can be taken out of the lab.

  1. Gantz VM, Jasinskiene N, Tatarenkova O, Fazekas A, Macias VM, Bier E, James AA. Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi. Proc Natl Acad Sci U S A. 2015 Dec 8; 112(49):E6736-43.
  2. Gantz VM, Bier E. Genome editing. The mutagenic chain reaction: a method for converting heterozygous to homozygous mutations. Science. 2015 Apr 24;348(6233):442-4.

CAT tails and tales of truncated protein synthesis

Just like any other man made machine, the natural protein synthesis machinery a.k.a. the magnificent ribosome, also fails at times. Stalling midway between its duty of forming peptide bonds between various amino acids, it produces truncated/nascent proteins that are unwanted by the cell. The cell has therefore developed a mechanism to get rid of these toxic truncated proteins. Shen et. al. [1] found that ribosome gets partially disassembled after stalling and also looses the mRNA, followed by recruitment of Ltn1p and Rqc2p to the subunit having the nascent peptide. Ltn1p binds to the side of the ribosome where proteins are spewed out and tags them with ubiquitin, a marker for destruction. Also, Rqc2p interacts with transfer RNA binding sites and stitches only alanine and threonine to the incomplete protein, producing a CAT tail (i.e. carboxy-terminal Ala and Thr extensions). CAT tails may induce heat-shock response to ensure degradation of the truncated proteins and also protects the cell from their toxic effects. In short, when the ribosome fails, the cell still manages to sustain protein synthesis even in the absence of any genetic instructions and targets them for recycling.

  1. Shen PS, Park J, Qin Y, Li X, Parsawar K, Larson MH, Cox J, Cheng Y, Lambowitz AM, Weissman JS, Brandman O, Frost A. Protein synthesis. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains. Science. 2015 Jan 2;347(6217):75-8.